In vitro diagnostic assays may be used to measure amounts of an analyte found in a body fluid sample or tissue sample. The analyte must be distinguished from other components found in the sample. Analytes may be distinguished from other sample components by reacting the analyte with a specific receptor for that analyte. Assays that utilize specific receptors to distinguish and quantify analytes are often called specific binding assays.
The most common receptors are antibodies and specific binding proteins such as Intrinsic Factor or Folate Binding Protein. Receptors are characterized by having a reversible specific binding affinity for an analyte or an analogue of that analyte. As used herein, an analogue generally is an analyte derivative carrying a detectable marker such as an enzyme, fluorescent molecule or other known label. The analogue is capable of binding to a receptor with about the same specificity and affinity as the analyte.
In heterogeneous specific binding assays described in the technical and patent literature, the receptor or other assay reagent of the specific binding reaction is often immobilized on a solid phase. Immobilization of the reagents is required to separate the bound components (for example an analyte bound to a receptor) from the unbound components.
The various methods by which a receptor or other reagent can be immobilized on a solid phase include adsorption, absorption or covalent bonding. However, many of the solid phase supports used in such assays are not inert and may sequester proteins and other substances from the sample by non-specific binding. Although glass is a relatively inert substrate, generally it has been found to be unsatisfactory for use in solid phase binding assays. See, for example U.S. Pat. No. 3,790,653 for a discussion of inadequacies of glass substrates.
Recently, however, procedures have been described for immobilizing an essentially soluble immunocomplex of a reagent and antiserum to the reagent on an inert glass fiber solid phase support. These procedures are disclosed in U.S. Pat. No. 4,517,288, incorporated by reference herein.
In these immunological immobilization procedures, soluble immunocomplexes are prepared by combining at least two immunochemically reactive substances with one another in solution. At least one of such immunochemically reactive materials is selected for its immunochemical specificity for an analyte of interest. For example, if the soluble immunocomplex is to be used in an immunoassay for the detection of thyroid stimulating hormone (TSH), then one component of the immunocomplex is selected for its immunochemical specificity for TSH. A typical example would be an antibody with specificity for TSH, i.e., an anti-TSH antibody. The second component of the immunocomplex could comprise an antibody preparation directed against the anti-TSH antibody. Antiserum to anti-analyte antibodies, for example to mouse anti-TSH antibodies, can be prepared by injecting purified mouse immunoglobulin G (IgG) into a host animal (i.e., goat), and thereafter harvesting the antiserum to the mouse IgG. The mouse anti-TSH antibody and the goat antiserum to mouse IgG are thereafter worked up as standard stock solutions.
Having prepared these stock solutions, a portion of each is combined in a buffered medium, The resulting immunocomplex, in an appropriate volume of buffer, may be spotted onto a delimited area of a glass fiber filter. Alternatively, the two components of the immunocomplex may be applied to the filter as separate buffered solutions and allowed to react in situ. In both instances, the point of application of the immunocomplex defines a reaction zone within the solid phase. The applied immunocomplexes become adsorbed and entrapped within the interstices of the beds of fibers within the glass fiber filter. The method of application can include dispensing of the immunocomplex solution with a manual or automated pipette, or with other automated equipment including assay analyzer instruments. Subsequent to application of the immunocomplex to the solid phase and the elapse of a suitable incubation period, the solid phase is dried under controlled conditions thereby yielding a stable reactive reagent which can be used in any one of a number of solid phase specific binding assay protocols.
Immunological immobilization, although useful in a variety of assay formats, has been noted to include a number of inherent disadvantages. Such factors as temperature, salt, pH and protein concentration have an influence on the formation of the double immunocomplex. These conditions can be difficult to optimize in order to effectively form the double immunocomplex. The presence of the additional immunoglobulins on the filter (e.g., antiserum to anti-analyte antibodies) can lead to nonspecific binding of proteinaceous and other biological materials. This can significantly decrease the assay sensitivity and overall performance. Moreover, given the inherent variability of IgG preparations from separate immunizations of the same or different host animals, lot-to-lot variability in titer, purity, specificity and affinity of IgG preparations must be accounted for in manufacturing procedures. Similarly, variability in the production of solid phase reagents may be encountered due to the tendency of immunocomplexes to become inhomogeneously distributed within stock solutions. That is, such immunocomplexes, while substantially soluble, may not remain completely soluble and may undergo some settling out of solution over time. Even with periodic mixing of stock solutions, gravitational influences, temperature gradients and other physical influences can cause subtle inhomogeneities within solutions applied to the solid phase reagents. These inhomogeneous solutions, resulting from protein aggregations, can cause blockage of the manufacturing lines used to spot the immunocomplex on the solid phase and other difficulties.
Some specific binding assays have been automated. A majority of the currently available automated assay systems, however, allow the detection of only one analyte in each cycle. Thus, in order to analyze more than one analyte in a single sample, one must wait for the testing cycle of a first analyte to be completed before a second analyte in the same sample can be tested and quantified. For most automated instruments a cycle time is usually at least 40minutes. Alternatively, the sample to be analyzed could be aliquoted into multiple testing samples and analyzed simultaneously to obtain the desired quantification information. This second approach not only requires a greater amount of the patient sample, but also necessitates additional capital expenditure in order to operate multiple analyzers simultaneously.
A limited number of random access enzyme immunoassay (EIA) systems utilize a unit dose concept in order to analyze more than one analyte in a given sample. In these systems, all the required reagents such as the immobilized antibody, antibody-enzyme or drug-enzyme conjugate and the substrate required for the generation of the signal, are packaged together in a single packet and each packet contains reagents sufficient for a single analysis. This single packet system is more expensive than bulk storage and packaging. The added expense reflects, for example, the cost of separately packaging each component required for an assay, specific requirements of the packaging material allowing maximum stability of the reagents, and the extra storage space needed for each unit dose. In addition, the time required for incubations at different steps of the assay causes the over-all time for generation of results to be longer for random access assay systems.